As much as half of the risk for atherosclerotic coronary heart disease (CHD) is genetic in nature, and an unprecedented amount of resources have recently been directed at association-based studies employing high density genome-wide scanning to identify genetic variation associated with CHD. These studies have identified a single region of the genome as the most highly associated with disease in an intergenic segment of chromosome 9 at p21.3, the Chromosome 9p21.3 CHD-Associated Region (C9CAR). The attributable risk for CHD explained by variation in this locus has been estimated at 10-15%. In addition to CHD, variants at C9CAR have been associated with other vascular diseases, including abdominal aortic aneurysm, stroke, and peripheral vascular disease. Although additional human genetics mapping efforts are underway in this laboratory and others, it is unlikely that they will identify the causative variation or elucidate the biology underlying the risk associated with 9p21.3 variants. Further progress will require efforts with basic molecular biology and animal model approaches. While there are several genes in this region of 9p21.3, none have been clearly linked to the disease-associated variation, and the causative variation has not been defined. Compelling recent evidence now suggests that a cyclin-dependent kinase inhibitor gene, CDKN2B, and a non-coding antisense RNA in the CDKN2B locus named ANRIL, are the most likely candidate genes linked to the variation at 9p21.3. Studies proposed here will identify which of these two candidates is the related gene, the mechanism by which 9p21.3 variation alters the function of this gene, and the fundamental disease-related pathways that it in turn regulates. Experiments in Specific Aim 1 will investigate allelic expression imbalance between the alleles of CDKN2B and ANRIL, and link this imbalance to variation in these genes as well as the intergenic C9CAR region. These studies will identify the causative gene and localize cis-acting transcriptional regulatory sequences. Additional experiments in this aim will characterize putative enhancer elements at C9CAR, identify the transcription factors that bind these elements and provide insights into cell types and signaling pathways that mediate the risk associated with this region. Specific Aim 2 will employ targeted deletion of CDKN2B and other genes at 9p21.3 in the apoE null atherosclerosis mouse model to provide insights into the cellular and molecular aspects of disease risk. Finally, in Specific Aim 3, targeted in vitro studies will further investigate how CDKN2B or ANRIL regulate basic cell fate decisions in vascular cell types and contribute to atherosclerotic disease. This work will identify the causative gene at 9p21.3, and disease related upstream and downstream pathways for further study and therapeutic targeting. PUBLIC HEALTH RELEVANCE: Significant expense and effort by groups of scientists around the world has led to identification of regions of the human genome that are associated with the genetic risk for various forms of cardiovascular disease. Additional research is required to understand the specific genes involved, and how they work to contribute to the disease process. Such information will allow the development of better therapeutics for these diseases.